Dihydrophenazine: a multifunctional new weapon that kills multidrug-resistant A cinet obact er baumannii and rest ores carbapenem and oxidative stress susceptibilities

Aims: T he current w ork aims to fully characteriz e a ne w antimicrobial agent against Acinetobacter baumannii , which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the e xtensiv e spread of resistance to almost all the a v ailable antimicrobials, the hunt for new antimicrobial agents is a high priority. Methods and results: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B f ollo w ed b y chemical str uct ure elucidation identified the active antimicrobial agent as 1-h y dro xy phenazine. Chemical synthesis yielded more deriv ativ es, including dih y drophenazine (DHP), which pro v ed to be the most potent against A. baumannii , yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP re v ealed multiple proteins with altered e xpression that could be correlated to the observ ed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to o xidativ e stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. Conclusions: T his w ork underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and noncon-ventional antimicrobial agent that is urgently needed.


Introduction
Acinetobacter baumannii is a multidrug-resistant (MDR) pathogen with stress-resistant capabilities (Gedefie et al. 2021 ).It is an opportunistic pathogen that represents a source of transferrable antibiotic resistance and virulence genes (Da Silva andDomingues 2016 , Nigro andHall 2016 ). A. baumannii is responsible for both hospital-and communityacquired infections (Pourhajibagher et al. 2016 ), including pneumonia, bloodstream infection, and meningitis that are associated with high mortality rates (Perovic et al. 2022, Yao et al. 2023 ).Unlike many other hospital-acquired bacteria, A. baumannii has developed a concerning ability to evade multiple antibiotics, making it a significant healthcare challenge (Chen et al. 2023 ).Carbapenems are considered among the last resorts for treating MDR A. baumannii (Li et al. 2006, Du et al. 2019, Palombo et al. 2023 ) but the frequent detection of carbapenem-resistant A. baumannii (CRAB), especially in developing countries like Egypt, has threatened their effectiveness (Zhu et al. 2022, Elwakil et al. 2023 ).Tigecycline and polymyxins are now considered the final lines of treatment for CRAB, but even these drugs are becoming less effective as resistant strains emerge (Chen et al. 2023, Sun et al. 2023 ).The situation gets more complicated by the fact that few new drugs are being developed to fight this pathogen.
In 2013, the Centers for Disease Control (CDC) listed A. baumannii as a MDR organism, with an alarming rate of acquiring resistance to new antimicrobials (Centers for Disease Control and Prevention (U.S.); National Center for Emerging Zoonotic and Infectious Diseases (U.S.); National Center for HIV/AIDS 2013).As of 2019, the threat level of A. baumannii has been escalated to urgent due to its rising carbapenem resistance, and the lack of current antibiotics or antibiotics in development to treat these infections (Centers for Disease Control and Prevention (U.S.); National Center for Emerging Zoonotic and Infectious Diseases (U.S.); National Center for HIV/AIDS 2019).Furthermore, the World Health Organization (WHO) has determined that A. baumannii and other carbapenem-resistant infections, such as Pseudomonas aeruginosa and Enterobacteriaceae, should be given top priority in the research and development of novel antibiotics ((WHO) 2017 ).
Environmental microorganisms are a valuable source of various antimicrobial compounds with unique structural and functional properties that are yet to be discovered (Amaning Danquah et al. 2022 ).The term 'antibiotic' originates from the word antibiose, which was coined by Paul Vuillemin in 1890 to describe the antagonistic behavior of different microorganisms toward each other (Vuillemin 1889 ).Alexander Fleming isolated "penicillin" from the fungus Penicillium notatum (Fleming 1929 ) and Giuseppe Brotsu discovered cephalosporin C from Cephalosporium acremonium (Bo 2000 ).Recent studies have identified many biomolecules with diverse chemical structures that have shown effective antimicrobial properties against drug-resistant bacteria (Amaning Danquah et al. 2022, Devi et al. 2023 ).These molecules can also serve as precursors for the development of more potent and safe antimicrobials.
The Egyptian environment has great potential to be a source of valuable active ingredients and compounds due to its largely unexplored biological resources.In the current study, the antimicrobial activity of a bacterial isolate from the Egyptian soil against A. baumannii was characterized, where 1hydroxy phenazine was identified as the active compound.Chemical synthesis generated other phenazine derivatives, including dihydrophenazine (DHP), which proved to be a potent antimicrobial agent.Moreover, DHP proved to affect multiple targets within the microbial cells, rendering A. baumannii more susceptible to both carbapenems and oxidative stress.

Bacterial strains and culture conditions
A. baumannii AB5075 (Jacobs et al. 2014 ) and Staphylococcus aureus strain Newman (Duthie and Lorenz 1952 ) were used in the study.A bacterial strain (code: NHM-077B) was also isolated from the Greater Cairo area (latitude: 30.048025, longitude 31.356440) and tested.All microbial strains were stored in brain heart infusion broth (Biolife, Italy) containing 30% glycerol at −80 • C. When required, A. baumannii AB5075 was either cultured in Luria-Bertani (LB) Medium (Lennox) (Serva, Germany) with shaking at 180 rpm or streaked on LB agar (Serva, Germany) plates, and incubated overnight at 37 • C. S. aureus strain Newman and NHM-077B were routinely grown on tryptic soy agar (TSA) plates (Biolife, Italy) or cultured in tryptic soy broth (TSB) (Biolife, Italy) with shaking at 180 rpm and incubated overnight at 37 • C.

Detection of antimicrobial activity against A. baumannii AB5075
Overnight cultures of A. baumannii AB5075 and the tested soil isolate were prepared in TSB.Bacterial cultures were sub-sequently adjusted to an optical density at 600 nm (OD 600 ) of 0.1.A diluted suspension of A. baumannii AB5075 (1:1000 in molten TSA) was evenly spread onto Petri dishes.Subsequently, 10 μl aliquots of the soil isolate culture were spotted on the inoculated agar surface.The plates were incubated aerobically at 37 • C for 24 h.The presence of an inhibition zone around the bacterial spot was considered as an indication of antimicrobial activity.This screening was performed in triplicate.

Molecular identification of the promising bacterial soil isolate
The isolate (code: NHM-077B) underwent molecular identification via polymerase chain reaction (PCR) targeting a 16S rRNA fragment.Universal primers U3 (5 -A GTGCCA GCA GCCGCGGTAA-3 ) and U4 (5 -A GGCCCGGGAA CGT A TTCAC-3 ) (James 2010 ) were used.A. baumannii strain AB5075 was used as a positive control for the PCR reaction and nuclease-free water was used as a negative control.The product was purified using the Promega Wizard SV gel and PCR clean-up system (Promega, USA), according to the manufacturer's instructions, and subjected to Sanger sequencing by Macrogen (Seoul, South Korea).The sequence was analyzed for nucleotide similarities using BLASTn tool available from the National Center for Biotechnology Information (NCBI).The resulting sequence was deposited in the GenBank database.

Assessment of the extracellular nature of the active antimicrobial metabolite
The extracellular nature of the antimicrobial metabolite produced by NHM-077B was assessed following the method described by Gislin et al.(Gislin et al. 2018 ).Briefly, a TSA plate was divided into two halves, then 400 μl of an overnight culture of NHM-077B was streaked on the surface of one half and 400 μl of uninoculated TSB was streaked on the second half.The plate was incubated at 37 • C for 24 h.The resulting NHM-077B growth was removed from the plate surface by slight scraping.The viable cells remaining on the plate were then inactivated by exposing the inverted plate to a chloroform-soaked filter paper and placed in the lid for 15 min.The chloroform was subsequently allowed to evaporate by keeping the plate open at 37 • C for 15 min.A. baumannii AB5075 was then streaked over the two halves of the agar plate and incubated overnight at 37 • C. The plate was then inspected for A. baumannii AB5075 growth.

Bioguided fractionation of the NHM-077B culture supernatant
NHM-077B was grown in one litre of TSB in a two-liter flask and incubated at 37 • C with shaking at 180 rpm for 24 h.Then the culture was centrifuged at 3200 x g for 15 min and the supernatant was filtered using a 0.45 μm syringe filter.The filtrate was extracted with an equal volume of ethyl acetate and the organic layer was dehydrated using anhydrous sodium sulfate.The transparent organic layer was decanted.The extract was concentrated to < 15 ml using a rotary evaporator, dried in a vacuum oven at 42 • C, and the residue was dissolved in dimethyl sulfoxide (DMSO) at 100 mg ml −1 and stored at −20 • C (Rajan and Kannabiran 2014 ).
The antimicrobial activity of the extract in DMSO against A. baumannii AB5075 was assessed by spotting onto an inoc- ulated LB agar plate as described above and by determining its minimum inhibitory concentration (MIC), by the broth microdilution method (CLSI 2018 ), using a concentration range of 25-2000 μg ml −1 .The MIC was considered the lowest extract concentration that showed no visible growth.An equivalent amount of DMSO was used as a negative control.Both experiments were done in triplicate.
For further fractionation, one litre of the NHM-077B culture supernatant was extracted with dichloromethane (DCM) (3 × 500 ml) to obtain the DCM fraction.The remaining aqueous layer was then separately fractionated using a Diaion HP-20 column, eluting sequentially with distilled water, 50% methanol, and 100% methanol.All fractions (DCM, water, 50% methanol, and 100% methanol) were evaporated under vacuum, dried completely, redissolved in DMSO (100 mg ml −1 ), and stored at -20 • C. The antimicrobial activity of each fraction against A. baumannii AB5075 was then assessed as above.

Identification of the antimicrobial compound(s)
The active DCM fraction from the NHM-077B was subjected to several chromatographic procedures including silica gel column chromatography, RP-C18 flash chromatography, and high-performance liquid chromatography (HPLC).The compounds in the active fraction were separated by thinlayer chromatography (TLC) on silica gel 60 (70-230 μm, 2 × 20 cm), eluted using DCM with increasing amount of ethyl acetate till 98:2%v/v.The chromatograms were visualized under UV.The solvents used for the extraction and fractionation were all analytical grades.JEOL ECZ-R500 MHz FT-NMR Spectrometer instrument equipped with Roy-al™ probe 5 mm combined Broadband and Inverse probe (JEOL, Japan) was used for 1 H and 13 C-NMR analyses ( 1 H-500 MHz and 13 C-150 MHz) of the separated active compounds and the chemical shifts were given in δ value.Tetramethyl silane (TMS) was used as an internal standard.Agilent HPLC 1100 series, equipped with Diode Array Detector (DAD) (Agilent Technologies, UK) and Puriflash 4100 (Interchim, USA) with DAD were used for further separation and purification of the compounds.High-resolution mass analysis was conducted on a Bruker MAXIS II Q-ToF mass spectrometer coupled to an Agilent 1290 UHPLC system.Separation was achieved using a Phenomenex Kinetex XB-C18 (2.6 mm, 100 × 2.1 mm) column and the following LC gradient profile: 5% MeCN + 0.1% formic acid to 100% MeCN + 0.1% formic acid in 15 min at a flow rate of 0.1 ml min −1 .MS parameters were: mass range m/z 100-2000, capillary voltage 4.5 kV, nebulizer gas 4.0 bar, dry gas 9.0 l min −1 , and dry temperature of 250 • C. The activity of the purified antimicrobial compounds was determined by spotting and MIC determination, as described above.

Synthesis of phenazine analogues
The 2-hydroxy phenazine was synthesized using the one-pot reaction of benzene-1,2-diamine and 1,4-benzoquinone as described by Kour et al. (Kour H 2014et al. 2014 ).Photochemical synthesis of DHP was carried out as described by Hass and Zumbrunnen (Haas and Zumbrunnen 1981 ).The method involved irradiating 1 × 10 −3 mol l −1 of phenazine solution in 0.1 mol l −1 of H 2 SO 4 by a 500 W projector lamp for 7 h.DHP was produced as a green precipitate and was recovered by vacuum filtration.The remaining reaction media contained 1-hydroxy phenazine as a by-product, which was also purified.
The structures of all synthesized derivatives were confirmed using various spectroscopic techniques: high-resolution mass spectroscopy, 1 H, 13 C and 2D NMR spectroscopy, 1 H-1 H COSY NMR spectroscopy, as well as heteronuclear single quantum coherence (HSQC) NMR spectroscopy and heteronuclear multiple bond correlation (HMBC) NMR spectroscopy.All compounds were dissolved in DMSO at a final concentration of 100 mg ml −1 , aliquoted, and stored at −20 • C except for DHP which was stored as a solid at room temperature and dissolved before each experiment.The antimicrobial activity of the synthesized derivatives was evaluated by determining their MIC against A. baumannii AB5075.The MIC of DHP against S. aureus strain Newman was also assessed.

Characterization of the growth pattern of A. baumannii AB5075 in the presence of DHP
The growth pattern of A. baumannii AB5075 was monitored in the presence of subinhibitory concentrations of 30, 60, 90, and 120 μg ml −1 DHP, which correspond to 0.24, 0.48, 0.72, and 0.96 of the MIC, respectively.This was achieved by diluting A. baumannii AB5075 overnight culture (1:200) in LB broth containing DHP in the respective four concentrations.The cultures were incubated at 37 • C and 180 rpm and their OD 600 was measured hourly.The results were compared to bacterial cultures containing an equivalent amount of DMSO, as a control.
Cell viability was assessed by the sulforhodamine B assay (Vichai and Kirtikara 2006 ).Briefly, cells were treated with 100 μl of the medium containing DHP at various concentrations (0.1-1000 μg ml −1 ).After 24 h of drug exposure, cells were fixed with trichloroacetic acid, washed with distilled water and treated with sulforhodamine B. Plates were washed with 1% acetic acid and allowed to air-dry overnight.The protein-bound sulforhodamine B stain was dissolved with trisaminomethane, and the absorbance of the solution was measured at 540 nm using a FLUOstar Omega microplate reader (BMG LABTECH, Germany).

Elucidation of potential mechanism of action of DHP against A. baumannii AB5075
The effect on cellular morphology and ultra-structures The effect of DHP treatment on the A. baumannii AB5075 cells was visualized by transmission electron microscopy (TEM).An overnight culture of A. baumannii AB5075 was diluted 1:200 in LB broth containing 100 μg ml −1 of DHP or an equivalent volume of DMSO.After 3 h of incubation at 180 rpm and 37 • C, the cells were pelleted, washed twice Downloaded from https://academic.oup.com/jambio/article/doi/10.1093/jambio/lxae100/7646869 by Bell College user on 02 May 2024 with phosphate buffered saline (PBS) and the formed pellet was fixed in 2% glutaraldehyde and 1% osmium tetroxide.Afterward, the pellet was dehydrated in alcohol and fixated in epoxy resin.Ultra-thin slices were cut to a 75-90 nm thickness and stained with uranyl acetate and lead citrate.The samples were examined at 50 000 × magnification using a transmission electron microscope JEOL (JEM-1400 TEM) (Nakamura et al. 2021 ).
Changes in the A. baumannii proteome in response to DHP treatment Diluted cultures of A. baumannii AB5075 in the presence of 100 μg ml −1 DHP or DMSO were prepared as described in TEM and incubated for 6 h at 37 • C and 180 rpm.Four biological replicas were tested for each condition.The cells were pelleted by centrifugation at 12 000 x g for 2 min and washed three times with PBS.The proteins were extracted by cell lysis using a lysis solution (8 mol l −1 urea, 500 mmol l −1 Tris HCl, pH 8.5) with complete ULTRA Tablets, Mini, EASYpack protease inhibitor cocktail kit (Roche, Germany).Protein assays of the extracts were performed using Bicinchoninic acid (BCA) assay (Pierce, Rockford IL) at Å562 nm before digestion.
Thirty μg of cell protein lysate from each sample was subjected to in-solution digestion.Protein lysate was reduced with 5 mmol l −1 tris (2-carboxyethyl) phosphine (TCEP) for 30 min.Alkylation of cysteine residues was performed using 10 mmol l −1 iodoacetamide for 30 min in a dark area.Samples were diluted to a final concentration of 2 mol l −1 urea with 100 mmol l −1 Tris-HCl, pH 8.5 before digestion with trypsin.For endopeptidase digestion, modified porcine trypsin (Sigma, Germany) was added at 40:1 (protein: protease mass ratio) and incubated overnight in a thermo-shaker at 600 rpm at 37 • C. The digested peptide solution was acidified using 90% formic acid to a final pH of 2.0.The resultant tryptic peptide mixture was cleaned up using stage tip as discussed earlier (Enany et al. 2023 ).Peptides were assayed using the peptide BCA method (Pierce, Rockford IL) at Å562 nm prior to injection to be 1.5 μg 10 μl −1 .
For mass spectrometric analysis, Nano-LC MS/MS analysis using TripleTOF 5600 + (AB Sciex, Ontario, Canada) interfaced at the front end with Eksigent nanoLC 400 autosampler with Ekspert nanoLC 425 pump was used.Detailed LC and MS methods were performed as previously described (Enany et al. 2023 ).
Generated raw LC-MS/MS data in Wiff format was searched against A. baumannii AB5075 database (TrEMBL database containing 3839 protein entries) using the Pro-teinPilot™ Software (version 5.0.1.0).The identified peptides were assembled into a list of reliable protein identifications using Pro Group™ algorithm.Analysis was searched with Bias Correction and biological modifications as ID focus.The false discovery rate (FDR) was maintained at 1% of the protein level to ensure high-quality results.
Data preprocessing included probabilistic quotient normalization (PQN) against a reference sample, filtration was set to allow only one missing value out of four per group, imputation was performed by replacing missing values with median ± 0.1 per group, and finally, data were z scaled.Proteins were considered differentially expressed if their detection level had a fold change ≥2 or ≤0.5.
Differentially expressed proteins between both experimental groups, together with unique protein hits shown in the DHP-treated group were functionally annotated.Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( http://www.genome.jp/kegg/) were used to annotate the above-mentioned protein candidates.

Antibiotics
The effect of subinhibitory concentrations of DHP on the susceptibility of A. baumannii AB5075 to different antibiotics was assessed using the Kirby-Bauer disc diffusion method with minor modifications (CLSI 2015 ).An overnight culture of A. baumannii AB5075 was diluted to reach an OD 600 of 0.1 and streaked over the surface of an agar plate containing a subinhibitory concentration of DHP (62.5 μg ml −1 ).Agar plates containing an equivalent amount of DMSO were used as controls.The tested antibiotics were ampicillin 10 μg, cefepime 30 μg, ciprofloxacin 5 μg, cotrimoxazole (trimethoprim/sulfamethoxazole 1:19) 25 μg, doripenem 10 μg, doxycycline 30 μg, gentamicin 10 μg, imipenem 10 μg, levofloxacin 5 μg, meropenem 10 μg, piperacillin 100 μg and ticarcillin/clavulanate 30/75 μg.All antibiotic discs were from (Hi-Media Mumbai, India).The MIC of meropenem against A. baumannii AB5075 was determined in the absence and in the presence of different subinhibitory concentrations of DHP (18.75,31.25,37.5,62.5 and 75 μg ml −1 ), using the broth microdilution method (CLSI 2018 ).Meropenem was tested in a concentration range of 0.0625 to 32 μg ml −1 and the MIC was the lowest concentration of the meropenem that showed no visible growth.

Oxidative stress
The impact of the subinhibitory concentrations of DHP on the A. baumannii AB5075 susceptibility to hydrogen peroxideinduced oxidative stress was evaluated.The testing was performed as described under the evaluation of DHP effect on antibiotic susceptibility using LB agar plates containing 62.5 μg ml −1 DHP and discs containing increasing concentrations of hydrogen peroxide (0.00625%, 0.0125%, 0.025%, 0.05%, and 0.1%).Discs containing sterile water and LB agar plates containing DMSO were used as controls.At the end of the incubation period, the diameters of the zones of growth inhibition were measured and the areas of the zones were calculated.

Statistical analyses
The effect of DHP on the susceptibility to different hydrogen peroxide concentrations was compared using student's t -test performed by GraphPad Prism v9.The P values < 0.05 were considered significant.In the proteomics analysis, the nonparametric Mann-Whitney U test was used as the data were not normally distributed (as evidenced by Shapiro's Wilk test of normality).The Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed using FactoMineR package and MixOmics, respectively.Heatmap was drawn using the R pheatmap package while other plots were generated using the R ggplot2 package (Team 2016 ).

Soil-driven P. aeruginosa NHM-077B exhibits promising antimicrobial activity against A. baumannii AB5075
A bacterial isolate, designated NHM-077B, was recovered from Egyptian soil and demonstrated significant antimicrobial activity against the MDR pathogen A. baumannii strain AB5075.This was evidenced by a clear zone of growth inhibition measuring 12 mm surrounding the isolate spot on an inoculated agar plate (Fig. 1 a).Molecular identification based on 16S rRNA gene sequencing confirmed the isolate as P. aeruginosa .The 16S rRNA fragment sequence of strain NHM-077B has been deposited in GenBank under accession number PP177584.A phylogenetic tree for the identified isolate with the top 10 hits is presented in Fig. S1A .

Pseudomonas aeruginosa NHM-077B produces an extracellular, DCM-soluble antimicrobial metabolite
The growth of A. baumannii AB5075 was inhibited on the media on which P. aeruginosa NHM-077B was grown, then killed using CHCl 3 and grew normally on the uninoculated media ( Fig. S1B ).Upon testing the ethyl acetate extract of NHM-077B culture supernatant for antimicrobial activity, a zone of growth inhibition was obtained (Fig. 1 b).The MIC of this extract was determined to be 500 μg ml −1 .On the other hand, upon further fractionation with DCM and incubation with Diaion HP-20 resin then elution with different solvents, only the DCM fraction showed a zone of growth inhibition (Fig. 1 c), while the water, 50% methanol and 100% methanol fractions did not (Fig. 1 d-f).These results confirm that the active antimicrobial metabolite produced by P. aeruginosa NHM-077B is extracellular and soluble in DCM.

1-hydroxy phenazine is the active antimicrobial metabolite in NHM-077B culture supernatant
Upon analysis of the DCM fraction, a yellow orange powder was obtained and identified as 1-hydroxy phenazine (Fig. 2 a).This compound exhibited significant antimicrobial activity against A. baumannii AB5075, forming a clear zone of growth inhibition on an inoculated agar plate (Fig. 2 b) with MIC of 1 mg ml −1 .A minor component identified as phenazine-1carboxylic acid was also detected with a MIC of 2 mg ml −1 .

S ynthesiz ed phenazine deri vati ves exhibit di ver se antimicrobial activities
DHP and 2-hydroxy phenazine were synthesized and 1hydroxy phenazine was retrieved during the synthesis processes.The MIC of the synthesized derivatives, DHP, 1hydroxy phenazine, and 2-hydroxy phenazine against A. bau-    matic area, while the high-resolution mass spectrum (Fig. 3 b) showed [M + H] + peak at m/z = 183.0915which confirms the molecular formula C 12 H 10 N 2 .Upon growing A. baumannii AB5075 in increasing sub-MIC concentrations of DHP, a dose-dependent inhibition was observed in the growth curves ( Fig. S2 ).

DHP exhibits a marginally safe cytotoxicity profile against human skin fibroblasts
The safety of DHP was evaluated by determination of the cytotoxicity against human skin fibroblasts.At 24 h of drug exposure, the IC 50 was found at 151.29 μg ml −1 ( Fig. S3 ).This indicated that DHP had a marginally safe profile toward the human dermal fibroblast cell line.

Changes in ultrastructure and morphology
The morphological effects of DHP treatment on A. baumannii AB5075 cells were visualized using TEM.The DMSO-treated control cells appeared elongated with intact well-defined cell walls and cell membranes and a densely stained cytoplasm (Fig. 4 a).On the other hand, the cells treated with 100 μg ml −1 DHP were slightly circular with a clear structural deformity and, disintegrating membrane, causing leakage out of the cellular components; they had a more relaxed DNA (Fig. 4 b).

DHP causes significant changes in the proteome fingerprint of A. baumannii AB5075
The A. baumannii AB5075 was treated with DHP for 6 h and compared with the DMSO-treated group as a control.A total of 412 proteins were detected in both the DHP-treated and control samples.Of which 61 (Tables 1 and S1 ) and 33 proteins (Tables 2 and S2 ) were identified only in the control and DHP-treated samples, respectively (Fig. 5 a).Interestingly, both experimental groups demonstrated remarkable segregation as indicated by the multivariate analysis (PCA) (Fig. 5 b) and the partial least squares-discriminant analysis (PLS-DA) ( Fig. S4 ).Some proteins were differentially expressed ( n = 24; fold change ≥2 or ≤0.5 (Table 3 ), with significant both Pvalue and FDR) with 7 upregulated and 17 downregulated proteins as shown in the heatmap (Fig. 5 c) and the volcano plot (Fig. 5 d).In addition, those significantly differentially expressed proteins with less strict cutoff fold change ≥1.5-1.99 or ≤0.7-0.501 are listed in Table S3 .
Overall, the functions of the proteins that were altered due to DHP-treatment belonged to different groups, including hydrolases, oxidoreductases, transferases, proteins involved in amino acid metabolic processes, transporters, proteins involved in t-RNA metabolic processes, proteins involved in DNA replication, proteins involved in RNA or DNA binding, proteins in lipid metabolic processes, proteins in the generation of precursor metabolites and energy, proteins involved in stress response, as well as proteins in carbohydrate metabolic processes.
For instance, DHP altered multiple oxidoreductases such as the glucose/quinate/shikimate dehydrogenase (QuiA), the ribonucleoside-diphosphate reductase (NrdB), the D-amino acid dehydrogenase (DadA2), and an amine oxidase.In addition, stress-related proteins included the universal stress protein UspA and a heat shock, GrpE family, protein.On the other hand, proteins involved in nucleic acids binding included the large ribosomal subunit protein uL23 (RplW), the large ribosomal subunit protein bL25 (RplY), the small ribosomal subunit protein bS20 (RpsT), the single-stranded DNA-binding protein (SSB), and the transcription antitermination protein (NusB).Also, the proteins altered by DHP and involved in the metabolic process included the ACP, which is involved in lipid metabolism, as well as, the glutamate-tRNA ligase and the leucine-tRNA ligase, which are involved in the tRNA metabolism.In addition, the oxidoreductase D-amino acid dehydrogenase (DadA2) is involved in amino acid metabolism.
The GO and KEGG pathway enrichment analysis were retrieved from both the unique proteins that appeared in the DHP-treated cells and the differentially significant proteins between both groups.As shown in Fig. S5 , most proteins were cytoplasmic proteins with binding capabilities.KEGG pathways highlight proteins involved in purine, pyrimidine, and thiamine activities.In addition, RNA polymerase and twocomponent system pathways were enriched.

DHP increases A. baumannii AB5075 susceptibility to carbapenems, cotrimoxazole, and oxidative stress
DHP enhanced the A. baumannii AB5075 susceptibility to cotrimoxazole, where a zone of growth inhibition with a mean diameter of 11 mm appeared on the inoculated agar plate containing 62.5 μg ml −1 DHP.In contrast, no inhibition zone was detectable on the control plate containing DMSO (Fig. 6 a).
Additionally, DHP enhanced the susceptibility of A. baumannii AB5075 to hydrogen peroxide-induced oxidative stress.No zones of growth inhibition were observed in the plates containing DMSO using 0.00625% and 0.0125% hydrogen peroxide discs whereas plates containing 62.5 μg ml −1 of DHP showed a mean zone diameter of 11 ± 0.5 and 17 ± 0.5 mm, respectively (Fig. 7 a).At higher hydrogen peroxide concentrations (0.025%, 0.05%, and 0.1%), the plates containing sub-inhibitory concentrations of DHP have pro- duced significantly ( P < 0.005) larger areas of growth inhibition compared to DMSO control plates (Fig. 7 b).

Discussion
The alarming rate of emergence of antimicrobial resistance in bacteria has made infectious diseases an urgent public health concern.The WHO announced that research and development of new antimicrobial drugs is urgently needed to combat carbapenem-resistant A. baumannii ((WHO) 2017 ).Microbial strains represent a rich unexplored source of antimicrobial compounds that can be utilized as lead molecules for developing more active antimicrobial compounds.
Here, a bacterial isolate from the Egyptian soil was evaluated as a producer of potential antimicrobial compounds against A. baumannii AB5075, a MDR strain (Jacobs et al. 2014 ).Isolate NHM-077B was identified as P. aeruginosa and demonstrated promising antimicrobial activity via an extracellular active metabolite.The antimicrobial activity of P. aeruginosa culture supernatant against A. baumannii was reported previously (Huang et al. 2022 ).Reports are also available about the antimicrobial activity of environmental P. aeruginosa strains against other problematic microbial species such as methicillin-and vancomycin-resistant S. aureus , Corynebacterium spp., Moraxella catarrhalis , Candida spp.(Xu et al. 2017 ), and Vibrio anguillarum (Zhang et al. 2017 ).
The major antimicrobial metabolite from P. aeruginosa NHM-077B was identified as 1-hydroxy phenazine.It is worth mentioning that the MIC of the isolated 1-hydroxy phenazine is higher than that of the whole NHM-077B culture supernatant extract; 1 vs. 0.5 mg ml −1 .A possible explanation for this observation could be that the extract contained other compounds that acted synergistically with the 1-hydroxy phenazine, and this was missing in the pure compound.Another explanation could be that the extract might contain an ingredient that enhanced the cellular uptake of the antimicrobial agent, and again this was missing in the isolated 1-hydroxy phenazine.Phenazine and its derivatives are wellknown pigmented secondary metabolites produced by Pseudomonas spp.and many other bacterial species as virulence factor with many effects on host cells (Pierson andPierson 2010 , Vilaplana andMarco 2020 ).They have a widely documented antifungal, antibacterial, and nematicidal properties (Kumar et al. 2005, Zhang et al. 2017, Nguyen et al. 2022 ).The production of the antimicrobial phenazine and its derivatives from Pseudomonas spp .has been reported previously, with phenazine-1-carboxamide being the most identified antimicrobial derivative (Peng et al. 2018, Biessy et al. 2021 ).To the best of our knowledge, this is the first report about the antimicrobial activity of a phenazine derivative against A. baumannii .The compound 1-hydroxy phenazine was reported as the most active P. aeruginosa metabolite against Aspergillus fumigatus , together with phenazine-1-carboxamide (Briard et al. 2015 ).It was also active against plant and fish pathogens (Liu et al. 2020, Qi et al. 2020 ).
Looking for a more active phenazine derivative, several compounds were chemically synthesized using phenazine as a lead molecule and their structures were confirmed.DHP was the most active among them with MIC of 125 μg ml −1 .In addition, DHP has a broad-spectrum activity by being also active against the Gram-positive S. aureus Newman strain with an MIC of 32 μg ml −1 .Moreover, DHP had a marginal safety profile against human skin fibroblasts, encouraging its potential therapeutic use with caution, at least for topical application.The result observed later, in which DHP at lower concentrations, with much lower toxicity to human cells, significantly sensitizes A. baumannii toward carbapenems, encourages its topical application as adjunctive therapy against CRAB while maintaining a safe dosage.In addition, lower concentrations of DHP rendered A. baumannii more sensitive to oxidative stress, which makes it more susceptible to killing by the host  immune system.Consequently, the proposal to use DHP for topical application minimizes systemic exposure and minimizes toxicity risks, especially since wound infections are one of the common and serious types of infections caused by this pathogen (Guerrero et al. 2010 ).Moreover, future research can be directed toward the introduction of modifications, either in structure or formulation, to maintain or enhance activity and reduce toxicity.
We then conducted a series of experiments to elucidate the mechanism of action and identify potential molecular targets of DHP against A. baumannii .TEM revealed that the cells became more circular, with leakage of cytoplasmic contents and the DNA appeared in some of the cells more relaxed.These observations are consistent with the reported effects of phenazine-1-carboxylic acid on V. anguillarum (Zhang et al. 2017 ), suggesting a potential shared mechanism of action.
Proteomics analysis showed that the level of many proteins involved in amino acid metabolism was abolished or reduced.For instance, DHP reduced the production of branched chain amino acid transferase, which catalyzes the formation of branched amino acids like valine and 3-methyl-2-oxobutanoate, the precursors of pantothenate and coenzyme A. Fatty acid elongation and biodegradation are also hypothesized to be halted by DHP that inhibits the production of the   ACP, which is the carrier of the growing fatty acid chain in fatty acid biosynthesis.The saturated chain fatty acids are essential components of the cell membrane in Gram-negative bacteria (Zhang and Rock 2008 ).
During the infection, bacteria are subjected to a variety of challenges imposed by the host's immune defenses.One type of stress faced during A. baumannii lung infection is reactive oxygen species such as the super oxide radical and hydrogen peroxide, which are produced by the innate immune system effector cells (Hampton et al. 1998, Juttukonda et al. 2019 ).The observed hypersensitivity to oxidative stress could be because DHP potentially depletes glutathione, a key  antioxidant defense molecule.This effect could be linked to DHP-mediated alterations in enzymes involved in glutathione metabolism, including a significant decrease in isocitrate dehydrogenase ( Table S3 ) and a significant increase in vitamin B12-dependent ribonucleotide reductase.In addition, DHP inhibits the level of adenosyl homocysteinase ( Table S3 ) and branched chain amino acid transferase, which are enzymes involved in the biosynthesis of L-cysteine.The availability of the amino acid precursors L-cysteine and L-glutamate and the activity of the rate-limiting enzyme glutamate cysteine ligase determine the rate of glutathione synthesis (Lu 2013 ).Moreover, DHP treatment reduced diaminobutyrate-2-oxoglutarate aminotransferase (Dat) to undetectable levels.This catalyzes the reversible reaction of 2-oxoglutarate with L-2,4-diaminobutanoate to form L-aspartate 4-semialdehyde and L-glutamate.The former is the precursor of spermidine necessary for the synthesis of pantothenate and CoA, as well as, glutathione (Ikai and Yamamoto 1997 ).
Interestingly, a recent study reported a correlation between exogenous glutathione availability and reduced meropenem susceptibility in A. baumannii (Yi et al. 2023 ).This raises the intriguing possibility that DHP's enhancement of meropenem activity, observed in our study, might be partially mediated by endogenous glutathione depletion and increased oxidative stress.Further research is needed to explore this potential connection.In addition, DHP increases the susceptibility of A. baumannii to hydrogen peroxide induced oxidative stress via the depletion of enzymes required in the synthesis of glutathione as well as both superoxide dismutase and thioredoxin peroxidase.Notably, superoxide dismutase inhibition was found to enhance A. baumannii susceptibility to oxidative stress (Steimbrüch et al. 2022 ) and inhibit the pathogenesis and virulence (Heindorf et al. 2014 ).
Phenazine-1-carboxylic acid was reported previously to reduce the tolerance of Xanthomonas oryzae , a rice bacterial pathogen, to oxidative stress.In accordance with our results, the proteomics analysis of the effect of phenazine-1-carboxylic acid on X. oryzae revealed a reduction in the protein level of ketol acid reductoisomerase and isocitrate dehydrogenase ( Table S3 ).In contrast with our results, the level of superoxide dismutase was not affected by phenazine-1-carboxylic acid in X. oryzae; however, the activity of superoxide dismutase was significantly reduced by phenazine-1-carboxylic acid treatment (Xu et al. 2015 ).This indicates that different antibacterial compounds might achieve similar outcomes through distinct mechanisms, highlighting the need for further investigation.
The level of many other enzymes and proteins associated with the tolerance to oxidative stress was reduced like oxidoreductases and universal stress proteins (Fiester and Actis 2013 ).The universal stress protein A (UspA) production was abolished entirely in the presence of DHP while the level of two other universal stress proteins was greatly reduced.UspA was found to have a role in tolerance to oxidative stress, low pH, and respiratory toxins (Elhosseiny et al. 2015 ).This finding is particularly significant considering UspA's established contribution to A. baumannii virulence in critical infections like pneumonia and sepsis (Elhosseiny et al. 2015 ).
DHP-treated cells also had lower levels of many lipoproteins which may influence cell membrane integrity; lipoproteins were reported to be upregulated in MDR A. baumannii strains (Wang et al. 2021 ).OmpA family protein (OmpA/MotB; type VI secretion system) level was also significantly reduced in DHP-treated samples ( Table S3 ).OmpA has a role in maintaining membrane integrity (Uppalapati et al. 2020 ) .The reduced membrane integrity can partially account for the visualized leakage of the cytoplasmic components out of the cell.
OXA-23 is the most prevalent mechanism of carbapenem resistance in A. baumannii (Jiang et al. 2022 ).DHP caused a decrease in the level of this carbapenemase enzyme; however, it was not statistically significant.Accordingly, we cannot directly link it to the observed increase in the susceptibility to carbapenems in DHP-treated cells.Nevertheless, considering the critical role of carbapenems as last-line antibiotics against numerous life-threatening pathogens, including A. baumannii (Li et al. 2006, Du et al. 2019, Palombo et al. 2023 ), restoring their efficacy against CRAB is of immense public health significance.Providing an effective therapeutic option in scenarios with limited treatment choices, DHP's ability to enhance carbapenem susceptibility represents an attractive solution worth further investigation in the fight against antimicrobial resistance.
Additionally, the reported proteomic changes in the presence of DHP pointed out a potential virulence modulation.The detectability of many virulence-related proteins was either abolished or reduced.DHP inhibited the production of pilin (a structural component of type IV pili) which is involved in the motility, adhesion, and biofilm formation of A. baumannii (Eijkelkamp et al. 2011, Harding et al. 2013 ).Some phenothiazine derivatives were reported to inhibit type IVmediated biofilm formation and motility in A. baumannii (Vo et al. 2023 ).Also, the level of the CsuC protein, involved in biofilm formation and attachment to abiotic surfaces (Harding et al. 2018 ), was reduced.Some proteins that are upregulated in A. baumannii under conditions mimicking a respiratory tract infection/colonization (Méndez et al. 2015 ), were downregulated in the presence of DHP such as the fimbria/pilus periplasmic chaperone (CsuC).On the other hand, some proteins that are downregulated during infection (Méndez et al. 2015 ), were upregulated by DHP such as the cysteine-tRNA ligase and aconitate hydratase B (Méndez et al. 2015 ).These proteins are involved in virulence and pathogenesis and represent possible targets for the development of antimicrobials against A. baumannii lung infection (Méndez et al. 2015 ).A summary of the potential major DHP cellular effects on A. baumannii is presented in Fig. 8 .
Collectively, the proteomics results show that DHP affects many targets inside A. baumannii ; any of which, or some combined, could be responsible for the observed inhibitory activity.This could occur through either compromising membrane integrity or inhibition of protein synthesis.Additionally, the results of the proteomics study suggest that DHP would enhance A. baumannii clearance in vivo by reducing the virulence and increasing the susceptibility to oxidative stress.Finally, DHP is a promising agent in restoring carbapenem susceptibility in CRAB.
In conclusion, DHP, a phenazine derivative, was synthesized based on the detection of 1-hydroxy phenazine as the active microbial metabolite from P. aeruginosa NHM-077B supernatant, a strain isolated from Egyptian soil.The developed DHP can be utilized as a broad-spectrum antimicrobial and can enhance the susceptibility of A. baumannii to carbapenems and oxidative stress.This molecule offers a new hope to overcome infections caused by A. baumannii by possibly disrupting multiple physiological processes within the cell, which could minimize the opportunity for the development of resistance.

Figure 1 .
Figure 1.The antimicrobial activity of P. aeruginosa NHM-077B culture and extracts against A. baumannii AB5075.Representative photos of agar plates inoculated with A. baumannii AB5075 and spotted with supernatant from the P. aeruginosa NHM-077B culture (a) and ethyl acetate extract of the culture supernatant (b), and DCM fraction (c).In addition to the water fraction (d), 50% methanol fraction (e), and 100% methanol fraction (f) of P. aeruginosa NHM-077B culture supernatant extract treated with Diaion HP-20 resin.The plates were incubated overnight at 37 • C and examined for growth inhibition.

Figure 2 .
Figure 2. Identification of 1-h y dro xy phenazine as the major antimicrobial compound produced by P. aeruginosa NHM-077B.(a) Chemical structure of 1-h y dro xy phenazine.(b) A photograph of the antimicrobial inhibition zone formed by spotting 1-hydroxy phenazine on an LB agar plate inoculated with A. baumannii AB5075.(c) 1 H-NMR spectrum chart of 1-h y dro xy phenazine.(d) Mass spectrum chart of 1-h y dro xy phenazine.

Figure 5 .
Figure 5. Analysis of the A. baumannii proteomes in DHP-treated cells.(a) A Venn diagram showing the proteins identified in each group uniquely and shared after the filtration process.(b) PCA of the DHP-treated samples (red) and the DMSO-treated ones (green).The analysis was performed with the FactoMineR package.(c) A heatmap representation of the proteins significantly differentially expressed between the DHP-and DMSO-treated samples.The map was generated using the R pheatmap package.(d) A volcano plot of all proteins significantly altered with log2-fold-change threshold = 1 and Benjamini-Hochberg corrected P -value threshold = 0.1.The plot was drawn using the R ggplot2 package.

Figure 7 .
Figure 7. Dih y drophenazine (DHP) enhances A. baumannii AB5075 susceptibility to o xidativ e stress.A gar plates containing 62.5 μg ml −1 of DHP or an equiv alent v olume of DMSO w ere streak ed with a diluted o v ernight culture of A. baumannii AB5075.(a) R epresentativ e photos of the plates containing discs impregnated with 0.0125% H 2 O 2 (b) The area of the zones of growth inhibition formed around discs containing increasing concentrations of H 2 O 2 placed on the surface of A. baumannii AB5075-inoculated agar plates containing 62.5 μg ml −1 DHP (gray bars) or equivalent amount of DMSO (black bars).* * * P < 0.005 and error bars represent the SD.

Table 1 .
List of top 22 proteins that were undetected in the DHP-treated samples.
*The proteins are ranked in descending order according to the abundance of the detected peptides in the DMSO-treated samples.
*The proteins are ranked in descending order according to the abundance of the detected peptides in the DHP-treated samples.